Conventionally, various kinds of animal and plant cells have been cultivated, and various modes of cultivation techniques have also been examined. The cell culture technique is a basic technique for use in many fields that deal with organisms. In particular, in the field of life science, the technique has become an indispensable technique for developing drugs and for clarifying pathologic mechanisms. In recent years, various examinations have been carried out not only on cell culture techniques for research purposes, but also on industrially productive culture methods for use in various fields, such as biology, medicine, pharmacy, immunology, etc. Moreover, in such a field as a medical field or the like, studies have been carried out in which organism cells are cultivated and the resulting cells are utilized as alternative organs, such as artificial organs, artificial teeth and bones, artificial skins, or the like.
In such cell cultivation, a cultivating process is normally carried out together with a culture solution serving as nutritive components in a predetermined container. The cell cultivation is mainly classified into two types depending on its natures, that is, one type in which the cultivation is carried out in a floating state in the culture solution and the other type in which the cultivation is carried out in an adhered state onto the bottom surface of the container. Most of animal cells have such adhesion dependence as to adhere to a substance and to be grown thereon, and in general, cannot survive for a long time in a floating state in vitro. For this reason, in order to cultivate cells having such adhesion dependence, a substrate that allows cells to adhere thereto is required.
As the cell culture substrates, petri dishes, flasks, multi-plates, or the like are generally used in the research laboratories. These instruments are mainly prepared as polystyrene molded products with surfaces that are subjected to a low-temperature plasma treatment, a corona discharge treatment or the like so as to provide hydrophilicity thereto, and these products are commercially available. In the case of anchorage dependent cells, these instruments are widely used for cultivation of fibroblasts, smooth muscle cells, vascular endothelial cells, corneal cells or the like, regardless of established cells and primary cells. Moreover, with respect to vasculature cells, these instruments are also widely used for so-called non-anchorage dependent floating cells that are established lymphatic corpuscles.
However, although growth of cells are observed on these cell culture instruments, the growth thereof is sometimes insufficient or the growth morphology of cells is disadvantageous in some cases depending on the kinds of cells. In particular, this defect is in particular conspicuous in the primary cultivation. Therefore, in many cases, the culture surface is coated with an extracellular matrix, such as collagen, gelatin or the like, or an adhesive agent such as fibronectin, laminine, vitronectin or the like, so as to improve the adhesion properties and growing properties of the cells, thereby solving the above-mentioned problems.
For example, as described in Non-Patent Documents 1, 2, 3, and 4 as well as in Patent Documents 1, 2 and 3, it has been known that, as the cell culture substrate, by using collagen, collagen gel, collagen sponge, a collagen sheet having cross-linked molecules to be formed into a three-dimensional structure, collagen sponge with through-holes formed therein, or the like, coated on a petri dish, human fibroblast cells or human cornification cells are disseminated and cultured thereon, so that a cultured epithelium/epidermis is produced, or a human cornification cell layer is formed on the upper surface of a human fibroblast cell so that a cultured mucous membrane/skin can be produced.
The collagen for use in preparing a collagen coat membrane is a type I collagen derived from animal connective tissues that are made soluble by applying an acid or enzyme thereto. This collagen is coated on a culture dish or the like, and then dried so that a collagen coat membrane is obtained. With respect to the collagen producing this cell culture substrate, for example, the collagen in connective tissues of bovine or swine is made soluble, and extracted collagen is used. However, in recent years, due to problems of BSE (Bovine Spongiform Encephalopathy), Aphtae epizooticae, or the like, the application of these to medical purposes has become difficult. Moreover, these materials derived from living organisms are easily denatured by chemicals such as alcohol, and putrefaction easily progresses therein, making it difficult to store for a long period of time. Furthermore, there have been strong demands for a cell culture method whose cell growth efficiency is further improved in comparison with a cell culture method in which these conventional cell culture substrates are used.
Moreover, in a cultivation of nervous-system cells, in many cases, the culture surface is coated with polylysine such as poly-D-lysine, poly-L-lysine, or the like, and the cultivating process is carried out. When coated with the polylysine, nervous-system cells are efficiently adhered, and in the case of established nervous-system cells, good growing morphology is preferably provided, with neurites being expanded in a high degree. Moreover, in a primary cultivation of brain cells of a rat fetus, neurites are desirably expanded, with a high degree of cell stabilization, so that a long-term cultivation can be carried out.
In this manner, although polylysine has desirable properties for cultivation of nervous-system cells, its defect is instability. That is, in the case when coated onto a general-use culture instrument as described above, the effect of polylysine is devitalized in two weeks when stored at room temperature, and in one month even when stored at 4° C. Moreover, this instability makes it difficult to carry out a sterilizing process after the coating process. For this reason, in order to use a culture instrument coated with polylysine, by using a time-consuming method in which a sterilizing operation has to be carried out on a culture instrument that has been preliminarily sterilized, polylysine needs to be coated thereon before use, and the resulting defect is that even when stored in a refrigerator, the usable period is limited only to about one month.
In this manner, in an attempt to coat the culture instrument with polylysine, problems arise in the stability and operability of polylysine, and in an attempt to sell the cell culture instrument preliminarily coated with polylysine on the market, high costs are required because of a number of jobs that have to be carried out under sterilized environments and difficulty in storing and managing the products after having been coated.
In view of these circumstances, as described in Patent Document 4, in order to solve the above-mentioned defects, the present inventors have proposed a cell culture substrate that has a structure containing a polymer having an amino group obtained by allowing a polymer containing an amino group capable of forming at least one Schiff base to react with a monomer unit of polyformyl paraxylylene.
However, the cell culture substrate produced by the aforementioned technique has a defect in that the reproducibility of cell adhesion is hardly obtained depending on conditions in which the Schiff base is made to react with polyformyl paraxylylene. Moreover, since the process of allowing the Schiff base to react after the coating process of polyparaxylylene is included, the number of processes increases, resulting in high costs.
Additionally, with respect to applied examples of polyparaxylylene, Patent Documents 5 and 6 disclose that to a product having a surface coated with polyparaxylylene, gigantic molecules such as protein are adsorbed so as to utilize the resulting product as a reaction container and that by utilizing its biological compatibility, this product is used as one portion of an intravital device for transporting insulin to a diabetic. Moreover, as described in Patent Documents 7 and 8, it has been known that a derivative membrane of polyparaxylylene having a surface with an amino group or an aminomethyl group formed thereon is utilized as a DNA chip-use binder.
In this case, as the roles of the substrate for use in cell cultivation, properties for allowing cells to adhere and for cultivating the cells are required. For this reason, Patent Document 6 has disclosed a technique in which the cell adhesion surface of an actual growing medium is coated with collagen so as to obtain an adhesion property. Moreover, general polyparaxylylene derivatives, such as polyparaxylylene, polymonochloroparaxylylene and polydichloroparaxylylene, as described in Patent Documents 5 and 6, have hardly any adhesion properties to cells, and are not suitable for the cell culture application.
In this manner, no cases have been reported in which a polyparaxylylene membrane, as it is, is utilized as a cell culture substrate, without being subjected to any chemical treatment, and in Patent Documents 7 and 8 as well, neither descriptions nor implications indicating that these derivatives are suitable for the cell culture application are found. In addition to these, as described earlier, the substrate needs to have high adhesion property to cells and it is essential for the substrate to adhere cells in a desirable adhesion morphology to be grown, and its required performances are completely different from those of techniques described in Patent Documents 7 and 8, in which cells are desirably joined to DNA probes so as to prevent them from being peeled off.